1. Field of the Invention
The invention relates to methods and systems for lipid production by oleaginous fungi and yeasts. In particular, sugars produced by pretreatment of biomass are separated from solid residues and used by oleaginous fungi or yeast to produce biomass and lipids, while solid residue is subjected to Simultaneous Saccharification (by cellulase and/or hemicellulase enzymes) and Fermentation (by oleaginous fungi or yeast) to also produce biomass and lipids.
2. Background of the Invention
The vast amount of lignocellulosic biomass such as crop straw remains underutilized in spite of great progress from decades of biofuel research and development efforts. At the same time, it is a daunting challenge for the nation to meet the 36 billion gallons per year biofuel production target by 2022 as mandated by the Renewable Fuel Standard. Producing “drop-in” biofuel has attracted great interest; however, various conversion routes suffer from lack of practical near-term options. Both significant improvement to existing technology and novel strategies for developing new technical options are needed to make significant advancements. Lignocellulosic biomass which contains mainly cellulose, hemicelluloses and lignin are considered as ideal renewable carbon source and can be converted to fermentable sugars upon pretreatment and enzymatic hydrolysis for culture of heterotrophic oleaginous microbes, such as yeast and filamentous fungi, to produce lipid. An effective pretreatment technology results in the disruption of cell wall physical barriers as well as cellulose crystallinity and association with lignin to enhance the accessibility of hydrolytic enzymes to biomass macrostructure, thereby increasing the yield of final products. Numerous pretreatment methods have been evaluated. In particular, pretreatment by dilute acid, with pH control, by ammonia, and with lime appear among the most promising options (Chang et al., 1997; Chang et al., 1998; Dale et al., 1985; Heitz et al., 1991; Holtzapple et al., 1991; Iyer et al., 1996; Mackie et al., 1985; Ramos et al., 1992; Saddler et al., 1993; Weil et al., 1998; Weil et al., 1997; Wyman et al., 2005; Yoon et al., 1995). Generally pretreatment processes mainly remove either hemicelluloses (eg. dilute sulfuric acid, hot water) or lignin (eg. lime, ammonia fiber explosion) as dissolved sugars or lignin-derived products in the pretreatment hydrolysate. Previous publications (Huang et al., 2009; Yeshitila Asteraye Tsigie et al., 2012; Yu et al., 2011) reported that the hydrolysate from dilute sulfuric acid pretreatment of lignocellulosic biomass could be effectively utilized by oleaginous yeasts. On the other hand, the remaining pretreated solid residues could be enzymatically hydrolyzed into monomeric sugars for further fermentation.
Another conversion process commonly applied in ethanol production is simultaneous saccharification and fermentation (SSF). SSF typically leads to higher product yields and requires lower amounts of enzyme because end-product inhibition from cellobiose and glucose formed during enzymatic hydrolysis is reduced (Emert & Katzen, 1980; Emert et al., 1980; Gauss et al., 1976; Spindler et al., 1989; Spindler et al., 1987; Takagi et al., 1977; Wright et al., 1987). Sugar released from cellulose during hydrolysis can be utilized in SSF. Combining SSF with hemicellulose sugar fermentation has attracted attention because of lower costs (Dien et al., 2000; Golias et al., 2002). The SSF process, however, has not been reported for oleaginous organism fermentation.
There are a number of challenges associated with application of the SSF process for lipid production using yeast or fungi: 1) the productivity and energy efficiency of lipid production are relatively low compared to bio-ethanol processes, 2) some toxic compounds that remain after enzymatic hydrolysis of pretreated lignocellulosic biomass may have an inhibitory effect on cell growth, 3) the sugar concentration in the enzymatic hydrolysates is not high enough to support the high cell density culture, 4) there may be a possibility that the cellulases used to hydrolyze the cellulose in the lignocellulosic biomass also have an adverse impact on some microorganisms that contain appreciable amounts of cellulose in their cell walls. SSF is often applied in cellulosic ethanol fermentation to decrease substrate inhibition and integrate the whole process, and the SSF process is also complicated by the fact that the optimum working temperature of most commercial cellulases is significantly higher than that of the microorganisms. Moreover, current SSF technologies designed for bio-ethanol production are not necessarily directly compatible with microbial lipid production. These strategies need be advanced and carefully integrated with the whole process of lipid accumulation.
Single cell oil (SCO) from microorganisms such as fungi and yeasts (Subramaniam et al., 2010) is considered as a potential feedstock for biofuel production because of advantages in high productivity. Some oleaginous microorganisms can produce lipids in amounts up to 70% of the total dry cell weight (Chen et al., 2009). However, the disadvantage of heterotrophic culture is that it requires an organic carbon source. Thus, many oleaginous yeasts have been studied for lipid accumulation on different substrates, such as industrial glycerol (Meesters et al., 1996; Papanikolaou & Aggelis, 2002), sewage sludge (Angerbauer et al., 2008), whey permeate (Akhtar et al., 1998; Ykema et al., 1988), sugar cane molasses (Alvarez et al., 1992) and rice straw hydrolysate (Huang et al., 2009). The use of non-starch biomass is critical and lignocelluloses with the advantages of its abundance and low cost can be used for organic carbon supply without concern for the “fuel versus food” issue. Since the fatty acid profile of microbial oils is quite similar to that of conventional vegetable oils, oleaginous yeast has been suggested as a favorable microorganism for a sustainable biodiesel industry (Zhao et al., 2010). In addition, previous reports indicated that temperature is an important factor in regulating the fatty acid composition in oleaginous fungi (Kendrick & Ratledge, 1992; Weinstein et al., 2000). Thermophilic fungi have the capability to grow in higher temperature, which is desirable for the SSF process because it would lead to higher cellulase activity.